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FITC1 Sigma

FluoroTag FITC Conjugation Kit

Synonym: FITC

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Description

Frequently Asked Questions

Frequently Asked Questions are available for this Product.

Analysis Note

Procedure
1. Dissolve protein and FITC in carbonate-bicarbonate buffer.
2. Slowly add FITC to protein with stirring. Cover with foil and stir 2 hours at room temperature.
3. Separate conjugate from free FITC on G-25 column. Collect fractions.
4. Pool fractions containing conjugate.
5. Determine F/P ratio of conjugate spectrophotometrically.
6. Stabilize with 1% bovine serum albumin and 0.1% sodium azide and store at 0-5 °C.

Features and Benefits

• Suitable for both small (1 mg) and large (5 mg) scale conjugations
• Completely aqueous procedure - no DMF needed
• Fast gel filtration separation of conjugate from excess FITC
• Complete protocols for conjugation and F/P ratio determination
• Sufficient reagents for at least 5 conjugations of 5 mg protein each and for optimization of F/P ratio before scale-up
• References for applications and protocols

Principle

Sigma offers a convenient kit for preparing FITC-labeled antibodies. Fluorescein isothiocyanate (FITC), Isomer 1, is a widely used fluorophore, popular because of its high quantum efficiency and stability when conjugated. FITC is yellow-orange in color with an absorption maximum at 495 nm. Upon excitation it emits a yellow-green color with an emission maximum at 525 nm. Conjugation occurs through free amino groups of proteins or peptides, forming a stable thiourea bond (see reaction). FITC conjugates of antibodies, lectins, hormones, and growth factors have been used in a variety of immunohistochemical and flow cytometry applications. The protocols have been optimized for antibodies, but may be adapted to other proteins by the end user.

Application

The Flourotag FITC Conjugation Kit can be used for Immunohistochemisty and Immunocytochemistry using Flow Cytometry. It is also be used for the conjugation of FITC to peptide hormones, cytokines, growth factors and proteins.

Legal Information

FluoroTag is a trademark of Sigma-Aldrich Co. LLC

Price and Availability


Amplified Detection

Kit component only

Description

Product #

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0.1 M Carbonate-bicarbonate buffer capsule, pH 9.0 5 capsules SDS    

Standard component

Description

Product #

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Fluorescein isothiocyanate isomer I, suitable for protein labeling, ≥90% (HPLC), powder 5 x 2 mg SDS F7250
Phosphate buffered saline, powder, pH 7.4, for preparing 1 L solutions 5 pkg SDS P3813
Sephadex G-25 column, 3.5 mL 1 SDS    
Sephadex G-25 column, 9.1 mL 1 SDS    
Safety & Documentation

Safety Information

Symbol 
GHS07  GHS07
Signal word 
Warning
Hazard statements 
Precautionary statements 
RIDADR 
NONH for all modes of transport
Protocols & Articles

Articles

Antibody Basics

Immunoglobulins (Igs) are produced by B lymphocytes and secreted into plasma. The Ig molecule in monomeric form is a glycoprotein with a molecular weight of approximately 150 kDa that is shaped more ...
Keywords: Affinity chromatography, Centrifugation, Chromatography, Digestions, Direct immunofluorescence, Gene expression, High performance liquid chromatography, Immunofluorescence, Ion Exchange, Microscopy, Precipitation, Purification, Rheumatology, Scanning electron microscopy

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Peer-Reviewed Papers
15

References

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